The widespread bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) is capable of carrying out the coupled translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (enzyme I and HPr) that are used for all sugars and sugar-specific proteins known as enzyme II. We are currently performing structure/function studies on E. coli enzyme I (MW=63,500) and its N-terminal domain (EIN, MW = 28,300), which is inactive in autophosphorylation but is phosphorylated at His 189 by transfer from phospho-HPr. The thermal stability of enzyme I and EIN in 10 mM phosphate, pH 7.5 was investigated by circular dichroism (CD) and differential scanning calorimetry (DSC). The DSC profile of EIN expressed in wild-type E. coli strain GI698 shows two endotherms at 50 and 57 degreesC in a first temperature scan and a conversion to the more stable form upon repeated scans from 10 to 70 degreesC. However, the overall secondary structure of EIN (as measured by far-UV CD spectra) remained essentially the same during heating cycles. Mass spectral analysis of unheated and heated EIN showed that the thermal conversion was due to the loss of covalently bound phosphate (80 Da mass). DSC of EIN expressed in a deletion strain of GI698 (without genes expressing HPr, enzyme I, and enzyme IIA/glc) showed a single two-state, reversible transition with a midpoint at 57 degreesC and an unfolding deltaH of approximately 140 kcal/mol. Moreover, DSC of intact enzyme I showed that phosphorylation of His 189 destabilizes a portion of the structure. After exhaustive dialysis of enzyme I against 10 mM pyruvate to remove covalently bound phosphate, only the endotherms for C- and N-terminal domains with Tm values of 41 and 54 degreesC, respectively, were resolved. Thus, phosphorylation of His 189 apparently produces a change in the conformation of enzyme I as well as in the isolated N-terminal domain (EIN) that may have physiological significance in the function of enzyme I in the PEP:sugar phosphotransferase system. Enzyme IIA/glc from Mycoplasma capricolum also has been expressed, purified, and characterized (Biochemistry 36, 6947-6953, 1997).